The present invention relates to a method and a reagent for measuring an amount of cholesterol in low density lipoproteins (hereinafter abbreviated as LDL) which is present in a living sample such as plasma and serum.
A lipid in plasma comprises mainly cholesterol, triglyceride, phospholipid, etc. and those plasma lipid is combined with apoprotein to form lipoproteins which is circulated in blood. The lipoproteins are classified, according to their density, into high density lipoproteins (HDL), LDL, very low density lipoproteins (VLDL) and chylomicrons (CM), etc. Among those lipoproteins, HDL has an action of transporting excess cholesterol stored in tissues to a liver, and thus has an action of preventing an arteriosclerosis, and on the other hand, LDL takes role as a main transporter for cholesterol from a liver to tissues and thus increase of LDL is considered as having close relation with arteriosclerosis.
Therefore, cholesterol in LDL (hereinafter abbreviated as LDL-cholesterol) has been considered to be a risk factor of arteriosclerosis and ischemic cardiopathy (coronary disease) and an amount of cholesterol in LDL is an important factor for diagnosis, remedy and prevention of those diseases.
As methods for measuring an amount of LDL-cholesterol, there have been known a precipitation method, an ultra-centrifugation method, an electrophoresis method, a calculation method based on a calculation equation, etc. Among those known methods, a precipitation method, an ultra-centrifugation method and an electrophoresis method require a pre-treating process for separation of LDL from other unnecessary lipoproteins by a precipitation/centrifugation treatment, an ultra-centrifugation treatment or an electrophoresis treatment, and thus they are accompanied with such problems that troublesome handling is required and it is not possible to conduct direct measurement with the use only of automatic analyzers which have widely been used in clinical tests at present.
Further a calculating method for calculating it from the total cholesterol amount, HDL-cholesterol amount and triglyceride amount, which has been known as Friedewald equation, has such problem that accurate measurement of an amount of LDL-cholesterol cannot be achieved when a sample containing triglyceride in an amount 500 mg/dl or more is used.
Recently, various methods have been developed in order to solve such problems as mentioned above in the known methods. For instance, a method disclosed in JP-A7-280812 (Japanese Patent Publication-Kokai-) is one example thereof. Namely, this method comprises agglutinating LDL with the use of an agglutinating agent and/or an antibody, introducing cholesterol in lipoproteins other than LDL into a separate reaction system which is not involved in the desired quantitative reaction, wherein cholesterol in lipoproteins other than LDL is consumed, then dissolving the agglutinated LDL with the use of a surfactant and/or an inorganic salt to such extent that quantitative analysis can be conducted, and finally subjecting LDL-cholesterol to quantitative analysis reaction and measuring an absorbance of the reaction solution.
However, this method is accompanied with such problem that it requires 3 or 4 kinds of reagents and thus can be applied only a limited kind of automatic analyzers which can conduct measurement with the use of 3 or 4 kinds of reagents, cannot be applied an automatic analyzers which can conduct only measurement with the use of reagents to the number of two. Further, there is such a problem in this method that reproducibility of measurement is decreased because of using 3 or 4 kinds of reagents.
Additionally, there has been such a method as conducting measurement without troublesome pre-treatment, which is disclosed in JP-A 58-165800 (Japanese Patent Publication-Kokai-). However, in this method, usable concentration range of a cholesterol esterase and a surfactant in the reagent is rather limited, and thus preparation of the reagent requires troublesome procedures, and further measuring conditions such as pH and intervals of measurement time have to be adjusted strictly, and in addition, only kinetic measurement of LDL-cholesterol, namely measurement by rate-assay method, can be conducted, because cholesterol also in HDL is involved in the reaction to some extent. For these reasons, this method is not said as practical.
Under the circumstances mentioned above, the problems to be dissolved by the present invention is to provide a method for measuring an amount of LDL-cholesterol in living samples directly with the use of widely used automatic analyzers without conducting troublesome pre-treatment for separation of LDL from unnecessary lipoproteins other than LDL which has been required in the known methods and also to provide a reagent usable in this method.